Detection and quantification of one or more nucleotide sequence target analytes in a sample using spatially localized target analyte replication

ABSTRACT

A target analyte which may be found in a substance, such as a biological or environmental substance, is assayed by inoculating a media with a sample of the substance. The target analyte is a unique nucleotide sequence of the RNA or DNA of a suspect organism, or any nucleotide target, or a combination of nucleotide sequences thereof, which organism may be found in the substance. The media contains selected target analyte amplifiers which will result in the amplification of any target analytes which are present in the substance. The media may also contain one or more labeled analyte-specific materials (LASMs) which can migrate through the media. The nature of the media is such that it will support target analyte-copying but will not allow the target analyte to migrate extensively within the media. After the substance to be assayed is added to the media and any target analytes are amplified, the LASMs will migrate by diffusion to resultant target analyte units or colonies in the media, thereby creating intensely labeled localized areas in the media which can be visually or mechanically detected. Each of the intensely labeled areas in the media will be surrounded by low intensity label halos. The number of high intensity labeled sites in the media will be proportional to the concentration of target analytes in the substance.

TECHNICAL FIELD

This invention relates to a method and paraphernalia for detecting thepresence or the absence of a target analyte in a substance sample; andquantifying the analyte when it is found to be present in the sample.The specimen being analyzed can be a biological substance; anenvironmental substance; a food stuff substance; or some other substancewhich can harbor the target analyte. The target analyte is a specificnucleotide sequence which is suspected of being in the substance beingtested. The presence or absence of the suspect organism or targetsequence can be determined by amplifying the target analyte with stranddisplacement amplification (SDA) or polymerase chain reaction (PCR)reagents. If a nucleotide sequence of RNA is the target analyte and SDAor PCR reagents are employed, it will be necessary to convert RNA to DNAby reverse transcription prior to the amplification step. Alternatively,target analyte RNA amplification methods, such as Qβ replicase or RNA-Xamplifications, may, in certain cases, be used without transcription.The analysis can be performed in a gel or a semi-solid media whichpermits amplification of the target analyte, but restricts migration ofthe amplified target analyte so that the products of amplification willform stationary detectable target analyte colonies in the media. Thecolonies may be detected and counted by various techniques, includingthe use of detectable SDA or PCR primers; or targetanalyte-complementary specific nucleotide sequences; or the use ofdetectable intercalating probes such as ethidium bromide, acridineorange, SYBR green, and related products. Such probes are available fromMolecular Probes, Inc. of Eugene, Oreg.

BACKGROUND ART

The analysis of specimens for the presence or absence of targetbacterial analytes, wherein the specimen is placed on a sterile growthmedia, is well known. Various bacteria can be detected in this manner inwater, food stuffs; and in sample biological specimens, such as urine,cerebrospinal fluid, pleural fluid, ascites, joint fluid, stool, and thelike. This testing procedure relies on the ability of the targetanalytes to replicate in the growth media to the extent that visiblecolonies of the target analytes can be observed. The various types ofbacteria can be differentially labeled so as to aid in thedifferentiation between different bacterial colonies which grow in themedia. When this analytic procedure is employed, the time necessary toform detectable colonies can be as short as one day, and as long asseveral weeks.

J. L. Burg et al describe a real time fluorescence detection procedureof RNA amplified by Qβ replicase, in Vol. 230 of Analytical Biochemistry(pp 263-272) 1995, Academic Press, Inc. The aforesaid detectionprocedure is non-specific and requires a considerable amount of samplepreparation in order to function properly. Additionally, the Burg et alprocedure cannot be used to detect DNA in a sample, and cannot detectmore than one RNA target at a time. Quantification of any amplified RNAis a function of the time delay between the beginning of the procedureand the presence of detectable fluorescence in the sample container.

It would be highly desirable to be able to detect the presence orabsence of one or more replicable target nucleotide sequence analytes ina single test, and quantify the target analytes in a short period oftime. It would likewise be desirable to be able to detect organismswhich cannot replicate in acellular systems, and to detect DNA genomesthought to be present in most life forms.

DISCLOSURE OF THE INVENTION

This invention relates to a method and paraphernalia for detecting thepresence or absence of one or more nucleotide targets in a sample.Detection of the nucleotide target is accomplished by amplifying aunique portion of the DNA or RNA sequence in a suitable viscous media soas to form relatively stationary colonies of the amplified nucleotidesequence in the media, and then detecting and counting any resultantnucleotide sequence colonies which may form in the media. The targetanalyte being detected is thus the amplified nucleotide sequences orsequences of the suspect life form. Diffusion of the formed targetanalyte colonies in the media is restricted relative to the diffusion ofmarker or probe reagents.

The media contains, or is combined with, target analyte-specific primersand suitable enzymes and reagents which will cause amplification of thespecified nucleotide sequence in the target analyte DNA or RNA throughthe use of strand displacement amplification (SDA); or through the useof polymerase chain reaction (PCR), or any other amplificationprocedures such as single primer or primeness 3SR, and in certain cases,Qβ replicase RNA amplification. In certain cases, other amplificationprocedures as described in PCR Technology: Principals and Amplificationsfor DNA Amplification, Ehrlich, H. A. ed. 1989, Stockton Press, NewYork, may be used in performance of this invention. The result is thecreation of localized high-density population colonies of the specifiednucleotide sequence in the media. Segments of non-target genomes whichare contained in the sample and are introduced into the media, and whichare not replicated by the amplification step, will not multiply, norwill they be detected. The analysis is performed in a media which mayalso contain, or be combined with one or more labeled analyte-specificmaterials (LASMs). The LASMs include a synthetic nucleotide sequencethat is complementary to a portion of the specific nucleotide sequenceforming the target analyte and which specific nucleotide sequence hasbeen amplified. The label may be a free dye or fluorophore, or one whichis attached to the complementary synthetic nucleotide sequence. TheLASMs are initially distributed homogeneously throughout the media. TheLASMs, following the laws of diffusion in the media, will migratethrough the media to the amplified nucleotide sequence colonies, andwill bind to these nucleotide sequences thereby differentiallyhighlighting the colonies relative to the remainder of the media. Lowintensity labeled halos will form around each colony due to localizedmigration of LASMs toward the colonies, and at the center of each halowill be a high intensity peak corresponding to the labeled colony. Anyunamplified molecules of DNA or RNA present on the media will benon-detectable by virtue of their low concentration, even if stained byacridine orange, ethidium bromide or other stains. In certain cases, thelabel may be attached to one or both of the oligonucleotide primers usedfor PCR or SDA amplification. In these cases, the primers will functionas the LASMs. The use of a kinetic study of signal intensity can nullout non-specific background signals such as may be caused by cellulardebris, since such non-specific background signals will notsignificantly increase with amplification.

When a specimen sample is being assayed for the presence or absence ofonly one target analyte, the colonies may be detected by non-specificstains for DNA or RNA such as acridine orange or ethidium bromide. Thereason for this is that these fluorophores are able to intercalatenon-specifically into any amplified nucleotide sequence, when such isproduced, and specificity is achieved by the specificity of theamplification primers without the need to link the fluorophore tocomplementary nucleotide sequences. In cases where the sample is beingassayed for the presence of more than one target analyte, then more thanone complementary nucleotide sequence may be linked to one or moredifferent fluorophores in order to highlight any amplified nucleotidesequence colonies which form in the media and to determine their type.

In performing the analysis of this invention, it is important that theamplified nucleotide sequence of the selected target analyte DNA or RNAbe of such a size, i.e., about 150 mer to about 500 mer, so that thecopies will be unable to migrate rapidly within the media, and thus willremain relatively stationary within the media during the course of theanalysis. It is likewise important that the selected LASMs be of such asize, i.e., about 18 mer to about 40 mer, so as to be sufficiently smallso that they will be able to diffuse more rapidly than the amplifiedtarget analyte sequences within the media, and yet sufficiently long soas to possess the necessary specificity to the amplified nucleotidesequences. Ideally, a LASM of about 18 mer may be used. Additionally,two or more LASMs of appropriate size which are directed againstdifferent nucleic acid sequences in the amplified target analyte may beused for enhanced specificity. The media must of course also be of sucha type that the amplification reagents including the probes can diffusewithin the media sufficiently rapidly to allow amplification to occurmore rapidly than the products of such amplification can diffuse awayfrom the colony.

The sample is prepared for analysis by first lysing all of its cellularand organism components so as to release into solution all of thegenetic material of the cellular and organism components of the sample,see: Maniatis, T., Fritsch, E. F., and Sambrook, J. (1987) MolecularCloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press. A knownvolume of nucleotide-rich aliquot of the lysed sample is then introducedonto the media and evenly spread out over the media. If necessary,diffusion of the target analyte into the media could be augmented byvertical electrophoresis, and/or by reducing its size prior to analysisby restriction digestion or mechanical shearing. The genetic material,DNA and/or RNA of the target component of the sample is thus exposed tothe amplifying materials in the media. A target nucleotide sequence onthe exposed genetic material is thus amplified to the extent thatcolonies of copies of the target analyte will be formed on the media.Since the size of the target analyte, and thus the size of the copies,is relatively large, the amplified target analyte colonies will remainrelatively stationary on the media. The LASMs, if not pre-incorporatedin the media, will be added to the media after the amplification stepand be relatively homogeneously distributed there over. The LASMs aresized so as to be able to migrate in or through the media. Therefore,the LASMs will then migrate to the target analyte colonies and will bindto the copies in the target analyte colonies. This binding will causethe colonies to emit a higher level of the label signal than surroundingareas in the media, and in fact, the immediately surrounding areas whichborder the colonies will emit a weakened label signal. The net resultwill be the formation of "bright" spots surrounded by "dim" halos in themedia. In the event that there is no target analyte present in thesample, then the media will retain a relatively even level of labelsignal emission, and will appear to be evenly "colored". Detection ofthe binding of the complementary nucleotides containing a fluorophore totheir respective target analytes may also be accomplished bytime-resolved fluorescence as described in U.S. Pat. No. 5,485,530,Lokowicz et al. If desired, the sample could be applied to the mediaprior to cell lysis, so that the localization of the target with respectto cellular nucleotides may be ascertained, i.e., to determine whetherthe target is intracellular or extracellular. If desired, the LASMs, aswell as the other reagents required for amplification, could beimpregnated onto a solid support such as a nylon membrane or paperstrip, and allowed to diffuse into the media by placing the membrane ontop of the media.

The assay can be completed within several hours after inoculating themedia with the sample by producing copies of the target analytes in themedia. Viruses, bacteria, fungi, mycoplasma, protozoa, or specificnucleotides present in any type of cell or specimen can be assayed byusing the specific nucleotide sequence-amplification and detectionmethod of this invention, and since one knows the volume of the specimensample added to the media, by counting the number of highlightedcolonies in the media, one can detect the amount of target analyte perunit volume of the specimen sample, thus enabling the securement of aquantitative analysis of the sample for target analyte.

A portion of the media will be inoculated with a control samplecontaining no known target nucleotides, but treated and prepared exactlyas the known sample. This portion of the media will serve as a negativecontrol. In the event that amplified labeled colonies form in thenegative control area of the media, one can presume contamination of themedia or the amplification or lysing reagents, or all, and the resultsof the assay are invalid. Another portion of the media can also besegregated and used as a positive control by applying a samplecontaining a known amount of the target analyte(s) therein when the testis performed. If the positive control area of the media fails to havethe appropriate number and type of colonies, then the amplificationand/or detection steps of the assay are not functioning properly, andthe assay is invalid.

It is therefore an object of this invention to provide a method andapparatus which can be used to assay a specimen sample for the presenceor absence of a target analyte.

It is another object of this invention to provide a method and apparatusof the character described wherein the sample is placed on a media whichis combined with a target analyte-specific labeled material that canmigrate within the media.

It is a further object of this invention to provide a method andapparatus of the character described wherein the target analyte is asignature nucleotide sequence or sequences derived from RNA or DNA of alife form.

It is an additional object of this invention to provide a method andapparatus of the character described wherein the target analyte isdetected by forming amplified colonies thereof, and by differentiallylabeling any amplified colonies which form in the media.

These and other objects and advantages of the invention will become morereadily apparent from the following detailed description of theinvention when taken in conjunction with the accompanying drawings, inwhich:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a plan view of a labeled target analyte-specific and growthmedia mixture formed in accordance with this invention;

FIGS. 2 and 3 are plan views similar to FIG. 1 but showing the formationof varying degrees of localized intensely labeled areas in the mixturedue to the presence of the target analyte in the sample being assayed;and

FIG. 4 is a pictographic trace of the signal intensity levels emanatingfrom the sample as the latter is scanned by an automatic signaldetection instrument.

DETAILED EXAMPLE FOR CARRYING OUT THE INVENTION

Referring now to FIG. 1, there is shown a plan view of a rectangularsection of a media, denoted generally by the numeral 2, which is adaptedfor performing the method of this invention. The media field as shown inFIG. 1 is evenly shaded so as to indicate an evenly distributed labelsignal emission which will be detected by the human eye or by a scanninginstrument either before the media has been inoculated with the sample;or after introduction and incubation of the sample, when there is notarget analyte present in the sample. The area 3 in the media 2 is anarea which will not be exposed to the sample being assayed, but whichcontains all of the reagents used in the test, and therefore serves as anegative control area. The area 5 in FIG. 1 is an area which alsocontains all of the reagents used in the test. The area 5 will not beexposed to the sample, but will be exposed to a positive controlsolution which contains a known concentration of the target analyte.FIGS. 2 and 3 are illustrative of the resulting change in the emissionpattern in the media 2 when the target analyte is present in the sample.FIG. 2 shows the result of a relatively low level of target analyte inthe sample which will produce a plurality of more highly labeledcolonies 4 surrounded by halos 6 of lower level label in the media 2.FIG. 3 shows the result when there is a relatively high level of targetanalyte in the sample. It will be noted from FIGS. 2 and 3 that one cancount the colonies 4, and since the volume of the sample inoculum andthe size of the field of media 2 is known, one can derive aconcentration of target analyte per unit volume by following theprocedure of this invention. It will also be noted from FIGS. 2 and 3that the positive control area 5 will have the same number of coloniesirrespective of the number of colonies which form in the remainder ofthe media 2.

FIG. 4 is a pictograph of the emission intensity from the label thatwill be detected by an auto-analyzing instrument as it performs a linearscan of the media. The trace 8 represents the emission level of themedia per se; the dips 10 represent the low levels of emission in thehalos 6; and the peak 12 represents the high level of emission in thecolonies 4.

The following are examples of suspect organism-specific target analyteswhich can be detected utilizing the technique of this invention, andreagents for use in detecting each target analyte.

EXAMPLE 1

One organism that can be detected and quantified using the technique ofthis invention is the HIV-1 virus. A target analyte gene for detectingHIV-1 is viral polymerase, whose function is the replication of theviral genome. (It is noted that the polymerase gene is only one ofseveral HIV-1 genes that could be detected with this method. In fact, aprotocol that simultaneously quantifies the concentrations of two HIV-1genes, for example, both polymerase and protease, would be susceptibleto very few false positive results since the polymerase-to-proteaseratio will be relatively constant.) A forward primer that can be used toamplify the viral polymerase gene using PCR is: SEQ. ID NO:1:

    GCACTTTAAATTTTCCCATTAGTCC;

and a reverse primer is: SEQ. ID NO:2:

    CCTGCGGGATGTGGTATTCC.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The complementarynucleotide sequence can be attached covalently to a fluorophore whichwill provide the necessary emission signal. In this case the LASM shouldlack a hydroxyl group on the 3' position of the nucleotide at the 3' endof the oligonucleotide to inhibit amplification of the product resultingfrom hybridization of one LASM with another. If a separate LASM is notused, then the fluorophore will be attached to the 5' terminus of one orboth of the oligonucleotide primers used for PCR or to one or more ofthe internucleoside linkages. All primers and LASMs should lack ahydroxyl group on the 3' position of the nucleotide at the 3' end of theoligonucleotide to inhibit amplification of the product resulting fromhybridization of one LASM with another. Suitable media includeacrylamide gels containing 2.5 to 5.0% acrylamide, and between 0.025 and2.5% bis-acrylamide; or 0.5% agarose; and standard and low-melt agarosegels containing 0.75 to 1.5% agarose. As noted above, the technique ofthis invention is optimized by using media compositions with low gelpercentages, for example about 4 to 5% for an acrylamide gel, and 2% orless for agarose.

A medium impregnated with the appropriate PCR reagents can be preparedas follows. A 100 ml wash solution consisting of 20 mM Tris, having a pHof about 8.0, in 50 mM EDTA; and a 10 ml storage solution consisting of2 mM Tris, having a pH of about 8.0, in 0.5 mM EDTA are prepared. Thegel is immersed in 5 ml of the wash solution for about 15 minutes atroom temperature. This step is repeated 5 times in fresh wash solution.The gel is then immersed in 5 ml of the storage solution containing PCRprimers, enzyme and dNTPs, and the like.

For amplification by the SDA method, related primers containingappropriate restriction sites will be used. For example, a forwardprimer that can be used to amplify the viral polymerase gene by SDA is:SEQ. ID NO:3:

    TTGAATAGTCGGTTACTTGTTGACACTCGGCACTTTAAAT;

and a reverse primer is: SEQ. ID NO:4:

    ACCGACTATTGTTGACACTGCCTGCGGGATGTGGTATTCC.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. Theoligonucleotide-based LASM should not be complementary to either theforward or reverse primers, but be complementary to a portion of thetarget molecule. This act will specifically focus the detectable agentat the molecular colony, and not to primers that may be freelydistributed throughout the media. The complementary nucleotide sequencecan be attached covalently to one or more fluorophores which willprovide the necessary emission signal(s). If a separate LASM is notused, then the fluorophore will be attached to the 3' terminus of one orboth of the oligonucleotide primers used for SDA, or to one or more ofthe internucleoside linkages.

A medium impregnated with the appropriate SDA reagents can be preparedas follows. A 100 ml wash solution consisting of 50 mM potassiumphosphate, having a pH of about 7.4 and 6 mM MgCl2; and a 10 ml storagesolution consisting of the wash solution plus SDA primers, anappropriate restriction enzyme, exo-Klenow polymerase and dNTPs, isprepared. The gel is immersed in 5 ml of the wash solution for about 15minutes at room temperature. This step is repeated 5 times in fresh washsolution. The gel is then immersed in 5 ml of the storage solution.

A LASM can be prepared as follows. For a dye-isothiocyanate label, thecomplementary nucleotide sequence strands will contain one or moremodified bases carrying an appended primary amine. The strand to belabeled is dissolved in 0.1 M sodium bicarbonate having a pH of 9.0, andthe dye-isothiocyanate is dissolved in dry DMF at a concentration of 10mg/ml. The dye solution is then added drop wise to the complementarynucleotide sequence solution with stirring and the reaction incubated atroom temperature for 12 hours. Free dye is then removed by gelfiltration and the dye-complementary nucleotide sequence conjugatepurified by gel electrophoresis or HPLC. For a dye-succinimidyl esterlabel, the complementary nucleotide sequence to be labeled is dissolvedin 0.1 M sodium phosphate, having a pH of 8.0, and the complementarynucleotide sequence-label conjugating reaction is performed at 4° C. tominimize hydrolysis of the succinimidyl ester label. Various dye-labeledDNAs can be prepared directly by chemical synthesis and are widely usedfor DNA sequencing.

The target analyte can be embedded in the media in the following manner.A suspension of approximately 2.5×10⁷ cells is prepared from a tissuehomogenate or culture, and washed thoroughly in tissue culture medium(no fetal calf serum) or isotonic saline. The mixture is thencentrifuged at 2,000 g for 20 minutes at 4° C., the supernatant isremoved, and the nucleic acid-containing pellet is drained thoroughly.The cells are re-suspended in 5 mL of 50 mM Tris (pH 8.0) and 50 mM EDTAand frozen at -20° C. To the frozen solution is added 0.5 mL of lysozymesolution (10 mg/mL lysozyme in 0.25 M Tris (pH 8.0)) and the mixtureincubated at 4° C. for 45 min. One milliliter of STEP solution (0.5%sodium dodecyl sulfate, 50 mM Tris (pH 7.5), 0.4 M EDTA, 1 mg/mLProteinase K (added immediately before use) is added and the solutionincubated at 50° C. for one hour. Six milliliters of buffered phenol isadded and the emulsion centrifuged at 1,000 g for 15 minutes. The upper,nucleic acid-containing layer is transferred to a separate tube and 0.1volume 3 M sodium acetate and 2 volumes absolute ethanol is added. Theprecipitate, containing DNA and RNA, is redissolved in 5 mL 50 mM Tris(pH 7.5) and 1 mM EDTA. If only cellular DNA is desired, 200 μg RNase Amay be added. The nucleic acid may be applied directly to the media, orit may first be treated with a restriction enzyme or sheared with aneedle to reduce its average size.

The target analyte-amplifying PCR reaction can be performed as follows.The gel, specimen sample, PCR and LASM mixture is heated to 95° C. for 3minutes, and then 30 cycles of heating to 95° C. for 45 seconds,annealing at 55° C. for 90 seconds, and extending at 72° C. for 1 minutefor every 750 nucleotides. For example, if one is amplifying a targetthat is 750 nucleotides long, that one would allow the extensionreaction to proceed for 1 minute; on the other hand, if the target were375 nucleotides long, the one would allow the extension to proceed for30 seconds. The reaction is finished with a 10 minute extension at 72°C. The media is then examined to determine the presence or absence oftarget analyte colonies in the media.

The target analyte-amplifying SDA reaction can be performed as follows.The gel, specimen sample, SDA and LASM mixture is incubated at 37° C.for between thirty minutes and four hours. The media is then examined todetermine the presence or absence of target analyte colonies in themedia.

EXAMPLE 2

Another target analyte that can be detected and quantified using thetechnique of this invention is M. tuberculosis. The target gene fordetecting M. tuberculosis is inhA whose function is fatty acidbiosynthesis. The forward primer for amplification of inhA by PCR isSEQ. ID NO:5:

    ACCCAATCGAATTGCCACACCCCG;

and the reverse primer is: SEQ. ID NO:6:

    GGCGCGCGAGGCCGGCAAGTACGG.

These primers will amplify a 279 base pair region of the inhA gene in M.tuberculosis. If a separate LASM is used, it will be complementary tosome region between the aforesaid forward or reverse primers. Theoligonucleotide-based LASM should not be complementary to either theforward or reverse primers, but be complementary to a portion of thetarget molecule. This act will specifically focus the detectable agentat the molecular colony, and not to primers that may be freelydistributed throughout the media. The media and LASM preparationprocedures are essentially the same as set forth in Example 1, as is theintroduction procedure.

For amplification of the inhA gene by SDA, a forward primer is SEQ. IDNO:7:

    AGTCGGTTACTTGTTGACACTCGACCCAATCGAATTGCCA;

and the reverse primer is SEQ. ID NO:8:

    ACCGACTATTGTTGACACTGCGCGAGGCCGGCAAGTACGG.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The media and LASMpreparation procedures are essentially the same as set forth in Example1, as is the introduction procedure.

EXAMPLE 3

A further organism that can be detected and quantified by using thetechnique of this invention is INH-resistant M. tuberculosis. The targetanalyte gene for detecting INH-resisistant M. tuberculosis is mutantinhA whose function is fatty acid biosynthesis. The forward primer foramplification of the mutant inhA gene by PCR is SEQ. ID NO:9:

    ACCCAATCGAATTGCCACACCCCG;

and the reverse primer is SEQ. ID NO:10:

    GCCGATCGATGAACCCCGGAGGTTCC.

These primers will amplify a 159 base pair region of the mutant inhAgene in INH-resistant M. tuberculosis. If a separate LASM is used, itwill be complementary to some region between the aforesaid forward orreverse primers. The oligonucleotide-based LASM should not becomplementary to either the forward or reverse primers, but becomplementary to a portion of the target molecule. This act willspecifically focus the detectable agent at the molecular colony, and notat primers that may be freely distributed throughout the media. Themedia and LASM preparation procedures are essentially as set forth inExample 1, as is the introduction procedure.

For amplification of the mutant inhA gene by SDA, a forward primer isSEQ. ID NO:11:

    AGTCGGTTACTTGTTGACACACCCAATCGAATTGCCACAC;

and the reverse primer is SEQ. ID NO:12:

    ACCGACTATTGTTGACACTGCGATGAACCCCGGAGGTTCC.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The media and LASMpreparation procedures are essentially the same as set forth in Example1, as is the introduction procedure.

EXAMPLE 4

An additional organism that can be detected and quantified using thetechnique of this invention is HBV. The target gene for detecting HBV ispX gene whose function is transcription. The forward primer foramplification of the pX gene by PCR is SEQ. ID NO:13:

    GGTGAAGCGAAGTGCACACGGACCGGC;

and the reverse primer is SEQ ID NO:14:

    GCTGGGGGAGGAGATTAGGTTAAAGG.

These primers will amplify a 196 base pair region of the pX gene in HBV.If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. Theoligonucleotide-based LASM should not be complementary to either theforward or reverse primers, but be complementary to a portion of thetarget molecule. This act will specifically focus the detectable agentat the molecular colony, and not at primers that may be freelydistributed throughout the media. The media and LASM preparationprocedures are essentially as set forth in Example 1, as is theintroduction procedure.

For amplification of the HBV pX gene by SDA, a forward primer is SEQ. IDNO:15:

    AGTCGGTTACTTGTTGACACGGTGAAGCGAAGTGCACACG;

and the reverse primer is SEQ. ID NO:16:

    ACCGACTATTGTTGACACTGGCTGGGGGAGGAGATTAG.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The media and LASMpreparation procedures are essentially the same as set forth in Example1, as is the introduction procedure.

EXAMPLE 5

Another organism that can be detected and quantified using the techniqueof this invention is Pneumocystis carinii. The target analyte gene fordetecting Pneumocystis carinii is mitochondrial rRNA whose function isprotein synthesis. The forward primer for amplification of thePneumocystis carinii mitochondrial rRNA gene is SEQ. ID NO:17:

    GATGGCTGTTTCCAAGCCCA;

and the reverse primer is SEQ. ID NO:18:

    GTGTACGTTGCAAAGTACTC.

These primers will amplify a 344 base pair region of the mitochondrialrRNA gene in Pneumocystis carinii. If a separate LASM is used, it willbe complementary to some region between the aforesaid forward or reverseprimers. The oligonucleotide-based LASM should not be complementary toeither the forward or reverse primers, but be complementary to a portionof the target molecule. This act will specifically focus the detectableagent at the molecular colony, and not at primers that may be freelydistributed throughout the media. The media and LASM preparationprocedures are essentially as set forth in Example 1, as is theintroduction procedure.

For amplification of the Pneumocystis carinii mitochondrial rRNA gene bySDA, a forward primer is SEQ ID NO:19:

    AGTCGGTTACTTGTTGACACGATGGCTGTTTCCAAGCCCA;

and the reverse primer is SEQ ID NO:19:

    ACGTGTACGTTGCAAAGTACGTGTACGTTGCAAAGTACTC.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The media and LASMpreparation procedures are essentially the same as set forth in Example1, as is the introduction procedure.

EXAMPLE 6

An additional organism that can be detected and quantified using thetechnique of this invention is methicillin-resistant S. aureus. Thetarget analyte gene for detecting methicillin-resistant S. aureus ismecA which is a penicillin binding protein. The forward primer foramplifying the mecA gene by PCR is SEQ. ID NO:21:

    AAAATCGATGGTAAAGGTTGGC;

and the reverse primer is SEQ. ID NO:22:

    AGTTCTGCAGTACCGGATTTGC.

These primers will amplify a 533 base pair region of the mecA gene. If aseparate LASM is used, it will be complementary to some region betweenthe aforesaid forward or reverse primers. The oligonucleotide-based LASMshould not be complementary to either the forward or reverse primers,but be complementary to a portion of the target molecule. This act willspecifically focus the detectable agent at the molecular colony, and notat primers that may be freely distributed throughout the media. Themedia and LASM preparation procedures are essentially as set forth inExample 1, as is the introduction procedure.

For amplification of the mecA gene by SDA, a forward primer is SEQ. IDNO:23:

    AGTCGGTTACTTGTTGACACAAAATCGATGGTAAAGGTTG;

and the reverse primer is SEQ. ID NO:24:

    GCTGGGGGAGGAGATTAGACAGTTCTGCAGTACCGGATTT.

If a separate LASM is used, it will be complementary to some regionbetween the aforesaid forward or reverse primers. The media and LASMpreparation procedures are essentially the same as set forth in Example1, as is the introduction procedure.

It will be appreciated that if a separate LASM is used, it can becomplementary to any region between the forward and reverse primers.Specimen samples which can be analyzed by means of this inventioninclude tissue, blood, biological fluids such as sputum for respiratorytarget analytes, stool for intestinal target analytes. Essentially thepresence or absence, and quantification of any life form, including butnot limited to, protozoa, bacteria, viruses, fungi, and the like can bedetected in essentially any specimen sample by using the technology ofthis invention. Likewise, different life forms, such as viruses andbacteria, to name two, can be detected and quantified in a single testwhich is performed on a common sample by following the precepts of thisinvention. Thus completely different causes of respiratory disease,diarrhea, genital disease, and many other diseases can be ascertained ina single test using the procedure of this invention.

Since many changes and variations of the disclosed embodiments of theinvention may be made without departing from the inventive concept, itis not intended to limit the invention otherwise than as required by theappended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - <160> NUMBER OF SEQ ID NOS: 24                                       - - <210> SEQ ID NO 1                                                        <211> LENGTH: 25                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HIV-1                                                    - - <400> SEQUENCE: 1                                                         - - gcactttaaa ttttcccatt agtcc          - #                  - #                  25                                                                       - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HIV-1                                                    - - <400> SEQUENCE: 2                                                         - - cctgcgggat gtggtattcc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HIV-1                                                    - - <400> SEQUENCE: 3                                                         - - ttgaatagtc ggttacttgt tgacactcgg cactttaaat     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HIV-1                                                    - - <400> SEQUENCE: 4                                                         - - accgactatt gttgacactg cctgcgggat gtggtattcc     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 5                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus                                            - - <400> SEQUENCE: 5                                                         - - acccaatcga attgccacac cccg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 6                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus                                            - - <400> SEQUENCE: 6                                                         - - ggcgcgcgag gccggcaagt acgg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 7                                                   <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus                                            - - <400> SEQUENCE: 7                                                         - - agtcggttac ttgttgacac tcgacccaat cgaattgcca     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 8                                                   <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus                                            - - <400> SEQUENCE: 8                                                         - - accgactatt gttgacactg cgcgaggccg gcaagtacgg     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 9                                                   <211> LENGTH: 24                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus (INH resistant - #)                        - - <400> SEQUENCE: 9                                                         - - acccaatcga attgccacac cccg          - #                  - #                    24                                                                      - -  - - <210> SEQ ID NO 10                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus (INH resistant - #)                        - - <400> SEQUENCE: 10                                                        - - gccgatcgat gaaccccgga ggttcc          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 11                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus (INH resistant - #)                        - - <400> SEQUENCE: 11                                                        - - agtcggttac ttgttgacac acccaatcga attgccacac     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 12                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: M. tubercle bacilus (INH resistant - #)                        - - <400> SEQUENCE: 12                                                        - - accgactatt gttgacactg cgatgaaccc cggaggttcc     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 13                                                  <211> LENGTH: 27                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HBV                                                      - - <400> SEQUENCE: 13                                                        - - ggtgaagcga agtgcacacg gaccggc          - #                  - #                 27                                                                      - -  - - <210> SEQ ID NO 14                                                  <211> LENGTH: 26                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HBV                                                      - - <400> SEQUENCE: 14                                                        - - gctgggggag gagattaggt taaagg          - #                  - #                  26                                                                      - -  - - <210> SEQ ID NO 15                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HBV                                                      - - <400> SEQUENCE: 15                                                        - - agtcggttac ttgttgacac ggtgaagcga agtgcacacg     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 16                                                  <211> LENGTH: 38                                                              <212> TYPE: DNA                                                               <213> ORGANISM: virus HBV                                                      - - <400> SEQUENCE: 16                                                        - - accgactatt gttgacactg gctgggggag gagattag      - #                      - #     38                                                                      - -  - - <210> SEQ ID NO 17                                                  <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Pneumocystis carinii                                           - - <400> SEQUENCE: 17                                                        - - gatggctgtt tccaagccca            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 18                                                  <211> LENGTH: 20                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Pneumocystis carinii                                           - - <400> SEQUENCE: 18                                                        - - gtgtacgttg caaagtactc            - #                  - #                      - # 20                                                                   - -  - - <210> SEQ ID NO 19                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Pneumocystis carinii                                           - - <400> SEQUENCE: 19                                                        - - agtcggttac ttgttgacac gatggctgtt tccaagccca     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 20                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Pneumocystis carinii                                           - - <400> SEQUENCE: 20                                                        - - acgtgtacgt tgcaaagtac gtgtacgttg caaagtactc     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 21                                                  <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: S. aureus (methicillin resistant)                              - - <400> SEQUENCE: 21                                                        - - aaaatcgatg gtaaaggttg gc           - #                  - #                     22                                                                      - -  - - <210> SEQ ID NO 22                                                  <211> LENGTH: 22                                                              <212> TYPE: DNA                                                               <213> ORGANISM: S. aureus (methicillin resistant)                              - - <400> SEQUENCE: 22                                                        - - agttctgcag taccggattt gc           - #                  - #                     22                                                                      - -  - - <210> SEQ ID NO 23                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: S. aureus (methicillin resistant)                              - - <400> SEQUENCE: 23                                                        - - agtcggttac ttgttgacac aaaatcgatg gtaaaggttg     - #                      - #    40                                                                      - -  - - <210> SEQ ID NO 24                                                  <211> LENGTH: 40                                                              <212> TYPE: DNA                                                               <213> ORGANISM: S. aureus (methicillin resistant)                              - - <400> SEQUENCE: 24                                                        - - gctgggggag gagattagac agttctgcag taccggattt     - #                      - #    40                                                                    __________________________________________________________________________

What is claimed is:
 1. An assembly for determining the presence orabsence of a target nucleotide sequence analyte hereinafter referred toas target analyte, in a specimen sample, said assembly comprising:a) amedia into which the specimen sample can be introduced, and which isoperable to support amplification of the target analyte to an extentneeded to produce colonies thereof while limiting migration of anyamplified target analyte colonies which are formed within the media; b)target analyte-specific amplification reagents essentially homogeneouslydistributed throughout said media, said amplification reagents beingoperable to selectively amplify and form colonies of any of the targetanalyte which may be present in the specimen sample; and c) a labeledanalyte-specific material (LASM) which will bind to said target analyte,said LASM being essentially homogeneously distributed throughout saidmedia and capable of migrating through said media to an extent necessaryto differentially highlight any amplified target analyte colonies whichmay be formed in said media.
 2. The assembly of claim 1 wherein saidLASM is sufficiently mobile in said media so as to be capable of forminghigh intensity labeled target analyte colony areas in said media, whichareas are surrounded by lower intensity labeled zones in said media. 3.The assembly of claim 1 wherein said LASM is an artificial targetanalyte-specific nucleotide sequence which is complementary to saidtarget analyte.
 4. The assembly of claim 1 wherein said LASM is anintercalating agent which can intercalate into said target analyte. 5.The assembly of claim 1 wherein said amplification reagents arepolymerase chain reaction reagents.
 6. The assembly of claim 1 whereinsaid amplification reagents are strand displacement amplificationreagents.
 7. An assembly for determining the presence or absence of twoor more different target nucleotide sequence analytes hereinafterreferred to as target analytes, in a specimen sample, said assemblycomprising:a) a media into which the specimen sample can be introduced,and which is operable to support amplification of the target analytes toan extent needed to produce colonies thereof while limiting migration ofany amplified target analyte colonies which are formed within the media;b) target analyte-specific amplification reagents essentiallyhomogeneously distributed throughout said media, said reagents beingoperable to selectively amplify and form colonies of any of the targetanalytes which may be contained in the specimen sample; and c) labeledanalyte-specific materials (LASMs) which will bind selectively to saidtarget analytes, said LASMs being essentially homogeneously distributedthroughout said media and differentially labeled and being capable ofmigrating through said media to an extent necessary to differentiallyhighlight any amplified target analyte colonies which may be formed insaid media whereby any colonies of one of the target analytes which formwill be distinguishable from any colonies of another of the targetanalytes which form in the media.
 8. The assembly of claim 7 whereinsaid LASMs are sufficiently mobile in said media so as to be capable offorming high intensity labeled target analyte colony areas in saidmedia, which areas are surrounded by lower intensity labeled zones insaid media.
 9. The assembly of claim 7 wherein said LASMs compriserespective differentially labeled artificial nucleotide sequences whichare complementary to respective sequences of said target analytes. 10.The assembly of claim 7 wherein said amplification reagents arepolymerase chain reaction reagents.
 11. The assembly of claim 7 whereinsaid amplification reagents are strand displacement amplificationreagents.
 12. A method for determining the presence or absence of atarget nucleotide sequence hereinafter referred to as target analyte, ina specimen sample, said method comprising the steps of:a) providing aspecimen sample to be analyzed; b) providing a testing media suitablefor specimen sample introduction, said testing media containing targetanalyte-specific amplification materials which are essentiallyhomogeneously distributed throughout said media; c) releasing any ofsaid target analyte from target organisms which may be contained in thespecimen sample; d) reacting any released target analyte with saidtarget analyte-amplification material in said media so as to formessentially immobile amplified colonies of said target analyte in saidmedia; and e) reacting any released target analyte with a labeledanalyte-specific material (LASM)₋₋ which is substantially homogeneouslydistributed throughout said testing media and which can migrate throughsaid testing media, said detectable substance being operable to bindwith amplified target analyte units in any formed target analytecolonies in said media so as to differentially highlight said targetanalyte colonies from the remainder of said media.
 13. The method ofclaim 12 wherein said LASM is essentially homogeneously dispersedthroughout said media prior to reaction with said amplified targetanalyte units.
 14. A method for determining the presence or absence oftwo or more target nucleotide sequences hereinafter referred to astarget analytes, in a specimen sample, said method comprising the stepsof:a) providing a specimen sample to be analyzed; b) providing a testingmedia suitable for specimen sample introduction, said testing mediacontaining target analyte-amplification materials which are essentiallyhomogeneously distributed throughout said media and are selectivelyspecific to each of said target analytes; c) releasing any of saidtarget analytes from target organisms which may be contained in thespecimen sample; d) reacting any released target analytes with saidtarget analyte-amplification materials in said media so as to formessentially immobile amplified colonies of said target analytes in saidmedia; and e) reacting any amplified target analyte units in saidcolonies with labeled analyte-specific materials (LASMs) which aresubstantially homogeneously distributed throughout said testing mediaand which can migrate through said testing media, said detectablesubstance being operable to selectively bind with any target analytes incolonies thereof which form in said media so as to differentiallyhighlight said formed target analyte colonies from the remainder of saidmedia, whereby any amplified target analyte colonies which consist ofone of said target analytes will be differentially highlighted fromamplified target analyte colonies which consist of another of saidtarget analytes.
 15. The method of claim 14 wherein said LASMs compriserespective differentially labeled artificial nucleotide sequences whichare complementary to respective sequences of said target analytes.
 16. Amethod for quantifying a target nucleotide sequence hereinafter referredto as target analyte, in a specimen sample, said method comprising thesteps of:a) providing a fixed volume of a specimen sample to beanalyzed; b) providing a testing media suitable for specimen sampleintroduction, said testing media containing target analyte-specificamplification materials which are essentially homogeneously; distributedthroughout said media; c) releasing any of said target analyte fromtarget life forms which may be contained in said fixed volume specimensample; d) reacting any released target analyte with said targetanalyte-specific amplification material in said media so as to formessentially immobile amplified colonies of said target analyte in saidmedia; e) reacting any amplified target analyte units with a labeledanalyte-specific material (LASM) which is substantially homogeneouslydistributed throughout said testing media and which can migrate throughsaid testing media, said detectable substance being operable to bindwith target analyte units in any formed target analyte colonies in saidmedia so as to form differentially highlighted target analyte colonyareas in said media; and f) counting the number of highlighted amplifiedtarget analyte colony areas in the media so as to obtain a targetanalyte-per-unit-volume sample concentration.
 17. The method of claim 16comprising the step of determining the percentage of amplified coloniesof nucleic acid sequences in the sample and/or the percentage of in situlysed organisms or cells in the sample that contain said target analyte.18. A method for quantifying two or more target nucleotide sequenceshereinafter referred to as target analytes, in a specimen sample, saidmethod comprising the steps of:a) providing a fixed volume of a specimensample to be analyzed; b) providing a testing media suitable forspecimen sample introduction, said testing media containing targetanalyte-specific amplification materials which are essentiallyhomogeneously distributed throughout said media; c) releasing any ofsaid target analytes from target life forms which may be contained insaid fixed volume specimen sample; d) reacting any released targetanalytes with said sequence-amplification materials in said media so asto form essentially immobile amplified colonies of said target analytesin said media; and e) reacting any amplified target analyte units insaid colonies with labeled analyte-specific materials (LASMs) which aresubstantially homogeneously distributed throughout said testing mediaand which can migrate through said testing media, said LASMs beingoperable to selectively bind with any amplified target analyte unitswhich form in said media so as to differentially highlight saidamplified target analyte colonies from the remainder of said media,whereby any amplified target analyte colonies which consist of one ofsaid target analytes will be differentially highlighted from amplifiedcolonies which consist of another of said target analytes; and f)counting the number of each of the differentially highlighted amplifiedtarget analyte colony areas in the media so as to obtain targetanalyte-per-unit-volume sample concentrations of each of the targetanalytes which appear in the media.
 19. A method for determining thepresence or absence of a target nucleotide sequence, hereinafterreferred to as target analyte, in a specimen sample, said methodcomprising the steps of:a) providing a specimen sample to be analyzed,said specimen sample being suspected of containing free target analyte;b) providing a testing media suitable for specimen sample introduction,said testing media containing target analyte-specific amplificationmaterials which are essentially homogeneously distributed throughoutsaid media; c) reacting any free target analyte with said targetanalyte-specific amplification material in said media so as to formessentially immobile amplified colonies of said target analyte in saidmedia; and e) reacting any target analyte with a labeledanalyte-specific material (LASM) which is substantially homogeneouslydistributed throughout said testing media and which can migrate throughsaid testing media, said LASM being operable to bind with amplifiedtarget analyte units in any formed target analyte colonies in said mediaso as to differentially highlight said target analyte colonies from theremainder of said media.
 20. A method for determining the presence orabsence of two or more target nucleotide sequences, hereinafter referredto as target analytes, in a specimen sample, said method comprising thesteps of:a) providing a specimen sample to be analyzed, said specimensample being suspected of containing free target analytes; b) providinga testing media suitable for specimen sample introduction, said testingmedia containing target analyte-amplification materials which areselectively specific to each of said target analytes which areessentially homogeneously distributed throughout said media; c) reactingany free target analytes with said target analyte-amplificationmaterials in said media so as to form essentially immobile amplifiedcolonies of said target analytes in said media; and d) reacting anyamplified target analyte units in said colonies with labeledanalyte-specific materials (LASMs) which are substantially homogeneouslydistributed throughout said testing media and which can migrate throughsaid testing media, said LASMs being operable to selectively bind withany target analytes in any colonies thereof which form in said media soas to differentially highlight said formed target analyte colonies fromthe remainder of said media, whereby any amplified target analytecolonies which consist of one of said target analytes will bedifferentially highlighted from amplified target analyte colonies whichconsist of another of said target analytes.
 21. A method for quantifyinga target nucleotide sequence hereinafter referred to as target analyte,in a specimen sample, said method comprising the steps of:a) providing aspecimen sample to be analyzed, said specimen sample being suspected ofcontaining free target analyte; b) providing a testing media suitablefor specimen sample introduction, said testing media containing targetanalyte-specific amplification materials which are essentuallyhomogeneously disribusted said media; c) reacting any free targetanalyte with said target analyte-specific amplification material in saidmedia so as to form essentially immobile amplified colonies of saidtarget analyte in said media; e) reacting any amplified target analyteunits with a labeled analyte-specific material (LASM) which issubstantially homogeneously distributed throughout said testing mediaand which can migrate through said testing media, said LASM beingoperable to bind with target analyte units in any formed target analytecolonies in said media so as to form differentially highlighted targetanalyte colony areas in said media; and f) counting the number ofhighlighted amplified target analyte colony areas in the media so as toobtain a target analyte-per-unit-volume sample concentration.
 22. Anassembly for determining the presence or absence of a target nucleotidesequence analyte hereinafter referred to as target analyte, in aspecimen sample, said assembly comprising:a) a media into which thespecimen sample can be introduced, and which is operable to supportamplification of the target analyte to an extent needed to producecolonies thereof while limiting migration of any amplified targetanalyte colonies which are formed within the media; b) targetanalyte-specific amplification reagents which are essentiallyhomogeneously distributed throughout said media, said amplificationreagents being operable to selectively amplify and form colonies of anyof the target analyte which may be present in the specimen sample; andc) a detectable substance which is essentially homogeneously distributedthroughout the media and which will bind to said target analyte, saidsubstance being capable of migrating through said media to an extentnecessary to differentially highlight any amplified target analytecolonies which may be formed in said media.
 23. A method for determiningthe presence or absence of a target nucleotide sequence hereinafterreferred to as target analyte, in a specimen sample, said methodcomprising the steps of:a) providing a specimen sample to be analyzed;b) providing a testing media suitable for specimen sample introduction,said testing media containing target analyte-specific amplificationmaterials which are essentially homogeneously distributed throughoutsaid media; c) releasing any of said target analyte from targetorganisms which may be contained in the specimen sample; d) reacting anyreleased target analyte with said target analyte-amplification materialin said media so as to form essentially immobile amplified colonies ofsaid target analyte in said media; and e) reacting any released targetanalyte with a detectable substance, which is substantiallyhomogeneously distributed throughout said testing media and which canmigrate through said testing media, said detectable substance beingoperable to bind with amplified target analyte units in any formedtarget analyte colonies in said media so as to differentially highlightsaid target analyte colonies from the remainder of said media.
 24. Amethod for determining the presence or absence of a target nucleotidesequence hereinafter referred to as target analyte, in a specimensample, said method comprising the steps of:a) providing a specimensample to be analyzed, said specimen sample being suspected ofcontaining free target analyte; b) providing a testing media suitablefor specimen sample introduction, said testing media containing targetanalyte-specific amplification materials which are essentiallyhomogeneously distributed throughout said media; c) reacting any freetarget analyte with said target analyte-specific amplification materialin said media so as to form essentially immobile amplified colonies ofsaid target analyte in said media; and e) reacting any target analytewith a detectable substance, which is substantially homogeneouslydistributed throughout said testing media and which can migrate throughsaid testing media, said detectable substance being operable to bindwith amplified target analyte units in any formed target analytecolonies in said media so as to differentially highlight said targetanalyte colonies from the remainder of said media.